Hematopoietic stem cells (HSC) are heavily studied adult stem cells. The ability of these cells to replenish hematopoiesis is exploited routinely to treat hematologic disease via bone marrow transplantation (BMT). However, many patients lack access to BMT because they lack suitable donors. Thus, an alternative source of clinically viable HSC would expand access to BMT for the treatment of disease. Pluripotent and embryonic stem cells (P/ESC) could potentially be coaxed to generate clinically viable HSC and thus represent one possible alternative source for donors. However, bona fide HSC do not spontaneously emerge during P/ESC differentiation and have never been robustly induced experimentally. ESC-HSC can emerge from murine ESC by manipulating homeobox gene expression (e.g. HoxB4 and Cdx4) during their differentiation. However, these cells have limited repopulating potential, do not faithfully reconstitute lymphoid cells, and display a cell surface phenotype reminiscent of mid-gestation hematopoietic progenitors. These data suggest that ESC- HSC derived in this manner suffer from a developmental block and that ESC could be coaxed to yield bone fide adult-like HSC with the appropriate molecular cues. Unfortunately, little is currently known about the key molecular regulators of HSC specification and developmental maturation. Thus, to identify novel molecular regulators of HSC development, we acquired the gene expression profiles of HSC and their precursors from mouse embryonic day 9 through adulthood using Affymetrix gene chip technology. Computational analyses of the resulting data implicates a myriad of novel genes as putative regulators of HSC ontogeny. The RNA- binding protein, Zfp36l1, was predicted by our analyses to regulate HSC specification in the aorta-gonads- mesonephros (AGM). This understudied gene is expressed in the AGM hemogenic endothelium and known to directly target the RNA transcripts of several established regulators of HSC function (i.e. VEGF, Stat5, Notch1). We propose to functionally evaluate a role for Zfp36l1 and other select gene candidates in HSC development according to the following aims: Specific Aim 1. Test the hypothesis that Zfp36l1 is required for definitive HSC specification. Towards this aim, we will assess the ability of ectopic Zfp36l1 to induce hematopoietic commitment in differentiating P/ESC and study mice in which this gene is conditionally deleted from the hematopoietic lineage using assays for in vitro and in vivo HSC function. Specific Aim 2. Functionally evaluate prioritized gene candidates via lentiviral delivery to HSC. Here, we will use lentivirus to delivery genes to enriched WBM HSC prior to their transplantation into lethally irradiated mice and assess the ability of these genes to impact HSC repopulating activity. For these studies, we will focus on 20 genes computationally predicted to function as transcriptional regulators of HSC specification. Via these aims, we will establish Zfp36l1 as a novel regulator of HSC specification and identify additional critical regulators of ths process. Each of these newly identified genes may ultimately serve as directors of P/ESC fate. PUBLIC HEALTH RELEVANCE: Hematopoietic stem cells, the source of all blood, often behave aberrantly in hematologic disease. By uncovering novel molecular regulators of their function and development, we can achieve a better understanding of their aberrant function and develop novel therapies to treat hematologic disease.